Growth and development require the correct temporal and spatial expression of many genes. This proposal addresses how the Nuclear Factor I (NFI) transcription factors regulate the expression of steroid hormone-dependent promoters. In transfected HeLa cells, NFI-C and NFI-X repress the mouse mammary tumor virus (MMTV) promoter, while NFI-A and NFI-B do not. Expression of the p300/CBP or SRC-1 coactivators alleviate this repression. NFI-C represses activation of the MMTV promoter by the glucocorticoid receptor (GR) but does not repress activation by the progesterone receptor (PR). To define the mechanism of this repression: Aim 1) We will characterize the differences between 2 NFI proteins and the 2 steroid hormone receptors that are essential for cell-type and promoter-specific repression by NFI-C. Since NFI-C but not NFI-B represses MMTV expression, we will produce chimeras between these two proteins to determine the precise domain required for repression. Similarly, since repression is seen of GR-induced but not PR-induced MMTV expression, chimeras between the GR and PR will be generated to assess the domain required for repression. Lastly, since the MMTV promoter is repressed by NFI-C but the NFIGRE promoter is not, we will make chimeras between these two GR-responsive promoters to determine the sites needed for NFI-C-repression. Aim 2) We will use the minimum repression domains from aim 1 to identify proteins essential for repression in vivo and to assess the mechanism of repression. Domains of NFI-C and GR that are essential for repression will be used in GST-pulldown and two- hybrid analyses to identify other interacting proteins needed for repression. Proteins identified will be tested for their ability to affect repression by NFI-C in vivo. Chromatin Immuno- Precipitation assays (ChIP) will be used to distinguish between models where the NFI-C repression domain interacts directly at the MMTV promoter vs. those where it functions through titration of coactivators or other proteins. We will also test the hypothesis that changes in protein occupancy at the MMTV promoter are important for repression by NFI-C. The long term goal of this study is to define the proteins essential for NFI modulation of steroid hormone-responsive promoters. These studies complement our ongoing work on the role of the NFI genes in development.